Gene expression at the level of mRNA production is regulated both at the initiation and termination of transcription. Termination factor Rho is a homohexameric protein that releases newly synthesized RNA from Escherichia coli transcription complexes. Rhos is thought to act through ATP-fueled, 5'->3' travel along nascent RNA. This travel is achieved by coordination of the RNA-dependent ATPase activity of Rho with RNA binding and release. Upon the arrival of Rho at the transcribing RNA polymerase, its continued travel unwinds the RNA-DNA helix of the transcription bubble, which disrupts the ternary transcription complex. The goal of the proposed work is the understanding the mechanism of directional travel by Rho along RNA. A fungal-developed anti-biotic, bicyclomycin, kills Gram-negative bacteria through Rho inhibition. A model in which Rho rolls along RNA as it hydrolyzes ATP predicts that ATO hydrolysis in the circularly arrayed Rho subunits will be sequential and in a fixed order. This prediction will be tested by using rapid mix/chemical quench techniques to examine pre-steady-state ATP hydrolysis kinetics. Other features of the kinetic mechanism will also be characterized, including determination of ligand binding order. Evidence for catalytic cooperativity among the active sites of Rho will be pursued and the requirements for cooperativity characterized. The rolling model also predicts that when RNA is bound to Rho, an unbound 5' region is the signal to Rho for ATP hydrolysis. This prediction will be tested by creating situations in which Rho has more or fewer than the usual number of signal RNA molecules bound. Each of these situations predicts changes in the pattern of ATP hydrolysis by Rho. This pattern will be monitored using rapid mix/chemical quench techniques and a combination of nucleotide binding with isotope partitioning experiments. Variant forms of Rho with characterized defects in RNA binding interactions will be used to clarify which of the two types of RNA binding sites on Rho interacts with the RNA signal for ATP hydrolysis. The proposed work will test and characterize the rolling model for ATP- dependent directional travel by Rho along RNA, which is the key to its transcription termination activity. The results will provide a better understanding of how the energy of ATP hydrolysis can be transduced to movement.